The long-term goal of this research is to explore the molecular mechanisms that bacteria use for cell-cell communication. Here we propose an integrated structural, chemical, and biological study of recently identified quorum sensing circuits in two related bacteria, Vibrio harveyi and Vibrio cholerae. To develop a molecular understanding of how quorum sensing signals (called autoinducers) are detected, and how sensory information is transduced to control behavior on a community-wide scale, we will carry out in-depth studies that combine synthetic organic chemistry, bacterial genetics, biochemistry, and x-ray crystallography. We will identify signaling agonists and antagonists to provide lead compounds for the development of antibacterial drugs designed to modulate quorum sensing. More generally, a longstanding problem in the bacterial signaling field is to understand how extracellular information is transduced into cells. The proposed studies will further our mechanistic understanding of transmembrane signal transduction via two-component sensor kinases, of which these quorum sensing receptors represent particularly tractable examples. The proposed aims build on significant progress during the first funding period, in which extensive structure/function studies led to a specific mechanistic model for signal transduction by the quorum sensing receptor LuxPQ. This mechanism differs fundamentally from the canonical mechanism based on studies of chemotaxis receptors. In the first aim, we will use molecular genetic approaches coupled with x-ray crystallography to test and extend our model. The second aim is to use organic synthesis and high-throughput screening to identify novel LuxPQ agonists and antagonists. Biochemical and structural studies will be used to characterize their mode of action. Aims 3 and 4 represent a new effort to characterize the molecular mechanisms underlying the dominant quorum sensing pathway in the human pathogen V. cholerae. In preliminary studies, we have determined the chemical structure of the relevant autoinducer, CAI-1. We have purified and crystallized the CAI-1 synthase CqsA, a pyridoxal phosphate enzyme, and in the third aim, we propose to determine its structure, identify its substrates, and characterize its enzymatic mechanism. In the fourth aim, we will combine genetic and chemical screens with x-ray crystallography to probe the molecular details of the interaction between CAI-1 and its cellular receptor. PUBLIC HEALTH RELEVANCE: Quorum sensing is a process of cell-cell communication that allows bacteria to collectively control processes including biofilm formation and the secretion of virulence factors. We propose to study quorum sensing in the major human pathogen, Vibrio cholerae, and to identify molecules that target quorum sensing to inhibit virulence.